Project Summary Hypercholesterolemia and vascular inflammation are among the strongest causative factors in the development of atherosclerosis and in the progression of early atherosclerotic lesions into advanced vulnerable plaques. However, it is yet unclear whether vascular cholesterol accumulation directly contributes to vascular inflammation and, specifically, to polarization of macrophages into inflammatory phenotypes. In this project, we will investigate two mechanisms, which contribute to cholesterol-mediated inflammatory responses by macrophages, and will determine their quantitative significance in the development of atherosclerosis in a mouse model and the translational potential of relevant new biomarkers and therapeutic approaches. In an atherogenic mechanism, oxidized cholesteryl esters (OxCE) induce dimerization of toll-like receptor-4 (TLR4), which in turn mediates inflammatory and atherogenic responses in macrophages. In an atheroprotective mechanism, we have identified the secreted apoA-I binding protein (AIBP) as a factor, which significantly improves HDL function by accelerating cholesterol efflux, resulting in the disruption of lipid raft-dependent TLR4 dimerization and reduced inflammatory responses in macrophages. We will test the hypotheses that OxCE promotes and AIBP inhibits inflammatory macrophage polarization, foam cell formation and atherosclerosis. We have generated a monoclonal antibody blocking OxCE, as well as systemic and macrophage-specific Apoa1bp-/- mice to test these hypotheses. In complementary experiments, we will use infusions of recombinant AIBP and/or an AIBP-expressing adeno-associated virus to achieve increased AIBP levels in plasma and expect to inhibit atherosclerosis progression. Importantly, synergistic studies with other PPG Projects will examine the relevance of OxCE and AIBP mechanisms to atherogenic or atheroprotective functions of different monocyte and T and B cell populations. To translate our findings into clinic, we will use plasma from Multi-Ethnic Study of Atherosclerosis (MESA) subjects, who have elevated coronary artery calcium or have experienced major adverse cardiovascular events, to evaluate diagnostic and prognostic value of new OxCE and AIBP biomarkers. These results will be integrated with other biomarkers identified by the PPG Investigators. To examine novel therapeutic approaches to restrain cholesterol-induced inflammation, we will test the hypothesis that raising AIBP in the low-AIBP plasma and blocking OxCE in the high-OxCE plasma found in MESA populations will reduce plasma's atherogenic effects on human macrophages. The proposed studies will enhance our mechanistic understanding of cholesterol-mediated inflammation in atherosclerosis and will advance new biomarker and therapeutic strategies for cardiovascular disease.